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Biol. Bull. 201: 277-278. (October 2001)
© 2001 Marine Biological Laboratory

Dye Coupling Evidence for Gap Junctions Between Supramedullary/Dorsal Neurons of the Cunner, Tautogolabrus adspersus

S. J. Zottoli, D. E. W. Arnolds, N. O. Asamoah, C. Chevez, S. N. Fuller, N. A. Hiza, J. E. Nierman and L. A. Taboada

Department of Biology, Williams College, Williamstown, Massachusetts 01267

Many teleost fish have neurons whose somata lie on the surface of the medulla oblongata (supramedullary neurons), the spinal cord (dorsal cells), or both. The somata of these neurons in the cunner, Tautogolabrus adspersus, are arranged in a single, median, longitudinal row, from the posterior end of the fissura rhomboidalis through the anterior portion of the spinal cord (1). Comparative physiological studies in the cunner and other teleost fish have indicated that these cells make electrotonic connections with one another (2). To test the hypothesis that gap junctions exist between these cells, we have injected individual supramedullary/dorsal cells with Lucifer yellow and looked for dye coupling with neighboring neurons.

Cunner, 7.3–10.5 cm in body length, were initially anesthetized in 0.03% ethyl-m-aminobenzoate; when respiration ceased, they were transferred to a holding chamber where a 0.015% solution of the anesthetic was passed through the mouth and over the gills of the fish. The caudal portion of the medulla oblongata and rostral spinal cord were exposed, and a microelectrode filled with Lucifer yellow (Sigma; 5% in distilled water) was inserted into the soma of only one neuron. Since the neurons exposed in this area of the central nervous system could either be in a supramedullary position or a spinal cord position, we will refer to the cells as supramedullary/dorsal neurons. The dye was iontophoresed (-10 nA of current was pulsed for 200 ms at a rate of 3/s) for about 1 h. About 1 h after injection the fish were perfused with 10% phosphate-buffered formalin. The brain and spinal cord were then dissected out, dehydrated, and cleared with methyl salicylate. The whole brain was viewed with a fluorescent microscope.

The somata of supramedullary/dorsal cells are visible on the surface of the brain with the aid of a dissecting microscope. In seven fish, a single soma was located and filled with dye. Lucifer yellow traveled from the filled cell to adjacent ones in three of the seven fish. One cell rostral and two cells caudal to the filled cell contained dye in two fish. In the third fish two cells rostral to the filled cell contained dye (Fig. 1). In the four cases where there was no apparent dye transfer between neurons, the intensity of the fill appeared similar to that of filled neurons in which dye coupling did occur. To control for the possibility that extracellular leakage of the dye might label neurons other than the one being filled, a single dorsal gill was penetrated with a dye-filled microelectrode in two fish. The electrode was withdrawn to just outside the cell membrane and Lucifer yellow was iontophoresed extracellularly for 1 h, the fish was perfused, and the brain processed as described above. No dye was localized to any cell.



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  		Figure 1. Lucifer yellow injection of a single supramedullary/dorsal cell in the cunner. The cell on the far right was iontophoretically filled with dye; after fixation, dehydration, and clearing, the whole brain was viewed with a fluorescent microscope. The soma of the filled neuron gives rise to a single process that extends ventrally and bifurcates near the bottom of the photomicrograph. Two other rostral somata (arrows) contain dye as well, providing support for the existence of gap junctions between these cells. This is a sagittal view of the brain with dorsal up and rostral to the left. Calibration bar = 100 µm.

 
The transfer of Lucifer yellow from one supramedullary/dorsal cell to others provides morphological evidence for the existence of gap junctions. The lack of dye coupling in four fish does not necessarily mean that gap junctions do not exist between supramedullary/dorsal cells in these fish. For example, there may be a wide distribution of sites of electrical coupling, or Lucifer yellow may not have crossed the gap junctions (3,4). When dye coupling occurred, the fall-off in dye concentration from the filled cell to adjacent neurons was large, so more distal neurons may be equally well coupled but not contain dye. The cunner has between 35 and 40 supramedullary/dorsal cells. Electrical coupling measurements will help determine the extent of coupling between this group of neurons. Supramedullary/dorsal cells in the cunner are sensitive to tactile stimulation (5). Our results predict that neurons that are electrotonically coupled will fire synchronously with sufficient tactile stimulation.

This work was supported in part by Howard Hughes Medical Institute and Essel Foundation grants to Williams College.

Literature Cited

  1. Sargent, P. E. 1899. Anat. Anz., 15:212–225.
  2. Bennett, M. V. L. 1960. Biol. Bull., 119:303.
  3. Murphy, A. D., R. D. Hadley, and S. B. Kater. 1983. J. Neurosci., 3:1422–1429.[Abstract]
  4. Peinado, A., R. Yuste, and L. C. Katz. 1993. Neuron, 10:103–114.[ISI][Medline]
  5. Zottoli, S. J., F. R. Akanki, N. A. Hiza, D. A. Ho-Sang, Jr., M. Motta, X. Tan, K. M. Watts, and E.-A. Seyfarth. 1999. Biol. Bull., 197:239–240.[ISI][Medline]



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D. E. W. Arnolds, S. J. Zottoli, C. E. Adams, S. M. Dineen, S. Fevrier, Y. Guo, and A. J. Pascal
Physiological Effects of Tricaine on the Supramedullary/Dorsal Neurons of the Cunner, Tautogolabrus adspersus
Biol. Bull., October 1, 2002; 203(2): 188 - 189.
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